The overall goal of this project is to develop a highly sensitive system for cancer diagnostics based on targeted imaging of tumor neovasculature. Formation of new blood vessels through processes of angiogenesis and vasculogenesis is common, unique, and one of the earliest features of primary tumors and metastatic lesions. Timely imaging of functional biomarkers in tumor neovasculature will provide formidable opportunities for cancer diagnostics and treatment. In this respect, endothelial cell specific receptors for vascular endothelial growth factor (VEGF), such as VEGFR-2 (KDR/Flk-1), are particularly attractive targets, because they are over-expressed in tumor but not normal vasculature. Although VEGF would be a natural choice for targeted imaging of tumor vasculature, development of VEGF-based diagnostics is severely hindered by inactivation of this fragile molecule upon conjugation of contrast agents. To solve this problem for VEGF and other fragile targeting proteins, we have recently engineered a novel 15- aa humanized tag for genetic fusion to recombinant proteins. This tag, named Hu(C4), contains an unpaired cysteine available for site-specific modification via standard SH-directed chemistries. In our preliminary experiments we found that bacterially expressed Hu(C4)-VEGF fusion protein is functionally active and retains activity after site-specific modification of Hu(C4) with bulky (approximately 56 kDa) construct. We hypothesize that conjugation of contrast agents to one or two Hu(C4)-tags in VEGF will not destroy its functional activity. In Phase I of this project we will test this hypothesis with two types of contrast agents. First, we will derivatize Hu(C4)-VEGF with a cyanine dye Cy5.5 for near-infrared optical imaging and test constructs in vitro and in vivo. If successful, Cy5.5-Hu(C4)-VEGF would become a powerful and commercially viable tool for research and development of anti-cancer therapeutics in animal models. Second, we will derivatize Hu(C4)-VEGF with a 99mTc chelator hydrazinonicotinamide (HYNIC) and test this construct in vitro. Finally, we will optimize production Hu(C4)-VEGF to the level sufficient for animal studies. Accomplishing these specific aims will determine feasibility of VEGF-based imaging. We expect that Cy5.5- Hu(C4)-VEGF would be ready for commercialization after completion of Phase I. In Phase II we will perform animal studies with HYNIC-Hu(C4)-VEGF, undertake formal toxicology studies for this construct, and develop GMPcompatible production of Hu(C4)-VEGF.